Multiwell-Screen
Manual
Impressum
Information in this document is subject to change without notice.
No part of this document may be reproduced or transmitted without the express written permission
of Multi Channel Systems MCS GmbH.
While every precaution has been taken in the preparation of this document, the publisher and the author assume
no responsibility for errors or omissions, or for damages resulting from the use of information contained in this
document or from the use of programs and source code that may accompany it. In no event shall the publisher and
the author be liable for any loss of profit or any other commercial damage caused or alleged to have been caused
directly or indirectly by this document.
© 2015 Multi Channel Systems MCS GmbH. All rights reserved.
Printed: 25. 08. 2015
Multi Channel Systems MCS GmbH
Aspenhaustraße 21
72770 Reutlingen
Germany
Fon +49-71 21-90 92 5 - 0
Fax +49-71 21-90 92 5 -11
[email protected]
www.multichannelsystems.com
Microsoft and Windows are registered trademarks of Microsoft Corporation. Products that are referred to in this
document may be either trademarks and/or registered trademarks of their respective holders and should be noted
as such. The publisher and the author make no claim to these trademark.
Table of Contents
Welcome to the Multiwell-MEA-System ....................................................................................... 4 Important Safety Advice .............................................................................................................5 Operator's Obligations ................................................................................................................6 Guarantee and Liability...............................................................................................................6 Hardware ....................................................................................................................................7 Multiwell-MEA Headstage .......................................................................................................7 Multiwell-MEA Interface Board ...............................................................................................8 Multiwell-MEA Well Plates ....................................................................................................11 Installing the Software ..............................................................................................................12 System Requirements .......................................................................................................12 Multiwell-Screen Software ......................................................................................................... 13 Cardio Application .................................................................................................................14 Main Menu .........................................................................................................................14 Control Section .....................................................................................................................18 Data Display Section.............................................................................................................33 Result Section .......................................................................................................................37 Settings Files ........................................................................................................................42 Result Files ...........................................................................................................................43 Control Section .....................................................................................................................46 Data Display Section.............................................................................................................53 Result Section .......................................................................................................................59 Replayer Mode .......................................................................................................................... 64 General Software Features ......................................................................................................68 MEA Filter Configuration ........................................................................................................... 70 Theoretical Background............................................................................................................73 Cardiac Action Potential........................................................................................................73 Theoretical Background of Spike Detection..........................................................................74 Technical Specifications ............................................................................................................ 75 Contact Information ..................................................................................................................76 Welcome to the Multiwell-MEA-System
The Multiwell-MEA-System is the newest member of the Multi Channel Systems MCS GmbH MEA product family.
Featuring 24-, 72- and 96-well plate format, it is the perfect tool for medium and high throughput electrophysiology.
The Multiwell-MEA-System includes high-quality, low-noise amplifiers, freely-programmable stimulators, and a
digital signal processor for individual analysis. The data is sampled at up to 50 kHz per channel (simultaneously on
all channels). Thus, the accuracy of your data is guaranteed, whether you record from cardiac or neuronal
samples.
The Multiwell-MEA-System consists of four components:
1. The interface board contains the USB 3.0 interface to data acquisition computer, analog and digital in- and
outputs for synchronization with other instruments, and a freely programmable digital signal processor.
2. The headstage includes 288 channel amplifiers, analog/digital conversion, integrated heating, integrated
stimulator, current and voltage stimulation, selectable via software. Any electrode can be used as stimulation
electrode. The sampling rate is up to 50 kHz per channel on all electrodes simultaneously.
3. Three types of well plates are available: 24 well plates with 12 electrodes each, 72 well plates with 4 electrodes
each (in 96-well plate format) and 96 well plates with 3 electrodes in each well. Additionally is a reference electrode
integrated in each well. The well plates are made from opaque epoxy substrate with gold electrodes or from
transparent glass substrate with either TiN or PEDOT/CNT electrodes.
4. The data acquisition and analysis software Multiwell-Screen and Multiwell-Analyzer are included in the system.
The software is used for online monitoring and recording, data are displayed in real-time. Depending on your
application, please run Multiwell-Screen software in "Cardio" or "Neuro" mode. A separate analysis software,
Multiwell-Analyzer, provides tools for detailed offline analysis.
Important Safety Advice
Warning: Make sure to read the following advice prior to installation or use of the device and the
software. If you do not fulfill all requirements stated below, this may lead to malfunctions or breakage
of connected hardware, or even fatal injuries.
Warning: Always obey the rules of local regulations and laws. Only qualified personnel should be
allowed to perform laboratory work. Work according to good laboratory practice to obtain best results
and to minimize risks.
The product has been built to the state of the art and in accordance with recognized safety engineering rules.
The device may only
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be used for its intended purpose;
be used when in a perfect condition.
Improper use could lead to serious, even fatal injuries to the user or third parties and damage to the device itself
or other material damage.
Warning: The device and the software are not intended for medical uses and must not be used on
humans.
Malfunctions which could impair safety should be rectified immediately.
High Voltage
Electrical cords must be properly laid and installed. The length and quality of the cords must be in accordance with
local provisions.
Only qualified technicians may work on the electrical system. It is essential that the accident prevention regulations
and those of the employers' liability associations are observed.
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Each time before starting up, make sure that the power supply agrees with the specifications of the product.
Check the power cord for damage each time the site is changed. Damaged power cords should be replaced
immediately and may never be reused.
Check the leads for damage. Damaged leads should be replaced immediately and may never be reused.
Do not try to insert anything sharp or metallic into the vents or the case.
Liquids may cause short circuits or other damage. Always keep the device and the power cords dry.
Do not handle it with wet hands.
Requirements for the Installation
Make sure that the device is not exposed to direct sunlight. Do not place anything on top of the device,
and do not place it on top of another heat producing device, so that the air can circulate freely.
Operator's Obligations
The operator is obliged to allow only persons to work on the device, who
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are familiar with the safety at work and accident prevention regulations and have been instructed how to use
the device;
are professionally qualified or have specialist knowledge and training and have received instruction in the
use of the device;
have read and understood the chapter on safety and the warning instructions in this manual and confirmed
this with their signature.
It must be monitored at regular intervals that the operating personnel are working safely. Personnel still undergoing
training may only work on the device under the supervision of an experienced person.
Guarantee and Liability
The general conditions of sale and delivery of Multi Channel Systems MCS GmbH always apply. They can be
found online at http://www.multichannelsystems.com/sites/multichannelsystems.com/files/documents/Terms and
Conditions.pdf
Multi Channel Systems MCS GmbH makes no guarantee as to the accuracy of any and all tests and data
generated by the use of the device or the software. It is up to the user to use good laboratory practice to establish
the validity of his / her findings.
Guarantee and liability claims in the event of injury or material damage are excluded when they are the result of
one of the following:
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Improper use of the device.
Improper installation, commissioning, operation or maintenance of the device.
Operating the device when the safety and protective devices are defective and/or inoperable.
Non-observance of the instructions in the manual with regard to transport, storage, installation,
commissioning, operation or maintenance of the device.
Unauthorized structural alterations to the device.
Unauthorized modifications to the system settings.
Inadequate monitoring of device components subject to wear.
Improperly executed and unauthorized repairs.
Unauthorized opening of the device or its components.
Catastrophic events due to the effect of foreign bodies or acts of God.
Hardware
The Multiwell-MEA-System consists of two devices, the interface board and the headstage. The third component
is the well plate.
Multiwell-MEA Headstage
Place the well plate into the designated area of the Multiwell headstage. Touch the left sensor button to close the
brackets of the well plate contacting unit, touch the right sensor button to open the brackets again.
Connect the headstage via SATAp cable to the interface board. Ground the headstage, if necessary. Please use
the provided cable for the connection between grounding socket on the rear panel of the headstage and the
external ground.
Multiwell-MEA Interface Board
Connect the headstage via eSATAp cable to the interface board. Connect the interface board via USB 3.0 to
the data acquisition computer. Connect the interface board to the power outlet. Ground the interface board if
necessary. Please use the ground socket on the rear panel of the IFB.
Front Panel
Two Status LEDs
The status LEDs indicate the link status of headstage 1 or headstage 2. They light up when the headstage is
connected to the interface board via eSATAp cable. LED 1 lights up, when connecting the headstage to connector
1 on the side panel of the IFB, LED 2 in on when using connector 2.
Important: Please use connector 1 on the side panel of the IFB and USB A on the rear panel of the IFB, otherwise
the headstage will not work.
Auxiliary Channels
Two reserve auxiliary channels are available for future use. They have no function at the moment.
Digital IN / OUT
A Digital IN / OUT for 16 digital in- and output bits is available (68-pin MCS standard connector) on the rear panel
of the interface board. On the front panel four digital IN and four digital OUT connectors bits are also accessible via
Lemo connector DIG IN bit 0 to bit 3 and DIG OUT bit 0 to bit 3. If access to more bits of the DIG IN / OUT channel
is required, it is necessary to connect a Di/o board with a 68-pin standard cable. This Di/o board is available as
optional accessory.
Power LED
The power LED near to the MCS logo on the front panel of the interface board should light up when the MultiwellMEA-System is "ON", and the device is connected to the power line. If not, please check the power source and
cabling.
Rear Panel
Toggle Switch On / Off
Toggle switch for turning the device on and off. The Multiwell-MEA-System is switched to status "ON" when the
toggle switch is switched to the left. The device is switched "OFF" when the toggle switch is switched to the right.
If the Multiwell-MEA-System is "ON", and the device is connected to the power line, the Power LED on the front
panel of the interface board should light up. If not, please check the power source and cabling.
Power IN
Connect the power supply unit here. This power supply powers both, the headstage and the interface board of the
Multiwell-MEA-System. The device needs 12 V and 2.5 A / 30 W.
Ground
If an additional ground connection is needed, you can connect this plug with an external ground using a standard
common jack (4 mm).
Digital IN / OUT
A Digital IN / OUT for 16 digital in- and output bits is available (68-pin MCS standard connector). Please read
chapter "Pin Layout" (Digital IN / OUT Connector) in the Appendix for more information about the pin layout of the
connector. The Digital IN / OUT connection accepts or generates standard TTL signals. TTL stands for TransistorTransistor Logic. A TTL pulse is defined as a digital signal for communication between two devices. A voltage
between 0 V and 0.8 V is considered as a logical state of 0 (LOW), and a voltage between 2 V and 5 V means 1
(HIGH).
The Digital OUT allows generating a digital signal with up to 16 bits and read it out, for example, by using a Digital
IN / OUT extension Di/o from Multi Channel Systems MCS GmbH. You can utilize this digital signal to control and
synchronize other devices with the Multiwell-MEA-System. Bit 0 to 3 of the Digital OUT are separated and
available as Lemo connector DIG OUT 0 to 3 on the front panel of the interface board. So if you need only one,
two, three or four bits of the digital signal, you don’t need the additional Di/o. Please read chapter "Pin Layout"
(Digital IN / OUT Connector) in the Appendix for more information. The Digital IN can be used to record additional
information from external devices as a 16 bit encoded number. The Digital IN is most often used to trigger
recordings with a TTL signal. The 16 bit digital input channels is a stream of 16 bit values. The state of each bit
(0 to 15) can be controlled separately. Standard TTL signals are accepted as input signals on the digital inputs.
Warning: A voltage that is higher than +5 Volts or lower than 0 Volts, that is, a negative voltage, applied to the
digital input would destroy the electronics. Make sure that you apply only TTL pulses (0 to 5 V) to the digital inputs.
Analog Channels
Eight Analog IN channels are available via 10 pin connector. Please read chapter "Pin Layout" (Analog IN
Connector) in the Appendix for more information. The additional analog inputs are intended for recording additional
information from external devices, for example, for recording patch clamp in parallel to the MEA recording, for
monitoring the temperature, or for fluidic control. Two of these channels (No 1 and 2) are available via Lemo
connectors on the rear panel of the interface board. You could also use the analog inputs for triggering, but please
note that the digital inputs DIG IN 0 to 3 are intended for accepting TTL pulses. Signals on the analog channels are
digitized and amplified with a gain factor of 2.
Analog Channels 1 and 2
Two of the eight analog channels described above (analog channel 1 and 2) are directly accessible via two Lemo
connectors.
DSP JTAG Connector
The DSP programming connector is used to program the digital signal processor DSP.
USB 3.0 Connector
The USB connector is used to transfer the amplified and digitized data from all data channels and the additional
digital and analog channels to any connected data acquisition computer via USB 3.0 high speed cable. The USB
cable has to be connected to the USB port labelled with "A" on the rear panel of the interface board.
Important: It is recommended to connect the USB 3.0 high speed cable direct to the USB 3.0 port of the computer.
Do not use an USB hub!
Audio OUT
To the Audio Out (3.5 mm phone jack) you can connect an audio system to make the electrical activity audible.
This audio output is real-time. Headphones or a speaker can be connected directly to the AUDIO OUT. Only two
channels at a time can be converted into sound (Stereo).
Side Panel
Sockets for connecting up the headstage via eSATAp cable. Please use the connector labelled with "1".
Multiwell-MEA Well Plates
Three different types of well plates are available: Well plates with 24 wells have 12 electrodes in each well and
96-well format plates with 96 available wells have 3 electrodes in each well. Additionally there is a reference
electrode integrated in each well.
The 72 active well design included in a 96-well plate allows using 4 electrodes in each well. The first and the last
row of the plate, A and H, have to be filled with buffer. Because row A and H do not contain electrodes, they are
ignored from recording and analysis, so unintended edge effects are avoided.
The well plates are made from opaque Epoxy substrate with gold electrodes or from transparent glass substrate
(available in 24 formats only) with either TiN or PEDOT/CNT electrodes. The gold electrodes with 100 μm diameter
are ideally suited for recordings and stimulation. Every single electrode can be selected for stimulation. The well
plates should be used as consumables.
96 connected wells with 3 electrodes per well in a 96-Epoxy well plate.
72 connected wells with 4 electrodes per well in a 96-Epoxy well plate.
24-Epoxy well plate with 12 electrodes in each well.
Installing the Software
System Requirements
Software: The following Microsoft Windows ® operating systems is required: 64-Bit Windows 8.1 (English and
German versions supported) with the NT file system. Other language versions may lead to software errors.
Important: The Multiwell-Screen software needs Windows 8.1 for data acquisition. The Multiwell-Analyzer software
needs Windows 7 or Windows 8.1
Hardware: Multiwell-MEA-System and a data acquisition computer.
Due to the amount of recorded data, a computer with low performance may lead to performance problems;
therefore, Multi Channel Systems recommends an up-to-date computer with a SSD data memory. Please contact
MCS or your local retailer for more information on recommended computer hardware specification. Please note
that there are sometimes hardware incompatibilities of the data acquisition system and computer components;
or that an inappropriate computer power supply may lead to artefact signals.
It is not recommended to run any applications in the background when using the Multiwell-Screen-System. Please
remove all applications from the Autostart folder.
Important: You need to have installed the latest data acquisition driver to operate the device, which is
automatically installed with Multiwell-Screen software. The installation may be invalid if the data acquisition does
not respond. Please contact Multi Channel Systems MCS GmbH or your local retailer in this case.
Recommended Operating System Settings
The following automatic services of the Windows operating system interfere with the data storage on the hard disk
and can lead to severe performance limits in Multiwell-Screen. These routines were designed for use on office
computers, but are not very useful for a data acquisition computer.
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Turn off "Screen Saver".
Turn off "Windows Indexing Service". Only important for data hard drive.
Manual management of automatic Windows Update.
Power Options: Power scheme: Never turn off monitor, hard disk and system standby.
It is not recommended to run any applications in the background when using Multiwell-Screen software.
Remove all applications from the "Autostart" folder.
Be careful when using a virus scanner.
Please check the system requirements before you install the software. MCS cannot guarantee that the software
works properly if these requirements are not fulfilled.
Important: Please make sure that you have full control over your computer as an administrator. Otherwise,
it is possible that the installed software does not work properly.
Multiwell-Screen Software
The software Multiwell-Screen is developed for cardio and neuro applications and facilitates data acquisition and
analysis. The software is used for online monitoring and recording.
To start the software, please click the icon or select it from the program menu.
If a firmware update is available and the Multiwell-MEA-System is switched on, the firmware will be updated
automatically.
Before your first start, the Multiwell-Screen offers the general software determination for cardio or neuro
applications. Select the desired application by clicking on the respective icon in the "Select your Application"
dialog. The selected mode will be stored as default. To change later from cardio to neuro application or vice versa,
please use the "Application" menu, option "Select Application".
Cardio Application
Main Menu
The main menu window is divided in three parallel sections: The left side contains control functions, the middle part
displays data in overview and single view and on the right side you see the current results. All windows are framed
by the menu bar above and the footer below, which holds information about the status of the hardware and / or the
file name.
Menu Bar
Application Menu
Menu to change the "Application" mode via first option "Select Application". The "Select your Application" dialog
appears. Menu to "Switch to Replayer Mode" for replaying files. Menu to save and load configurations and to save
and restore default configurations. Menu to "Load" and "Export Results". "Reset the Window Arrangements"
restores the factory defaults of the docking status of the windows. "Exit" the program.
"Load Results"
Immediately after starting the Multiwell-Screen software before inserting a new wellplate, it is possible to load
a result file for further analysis. The main menu is empty except for the result section. Change the analysis
parameter for a different analysis of the dose-response curve.
"Export Results"
To be free for further analysis of the results created with Multiwell-Screen, the software offers the possibility to
export results to ASCII format. The export file is organized as a comma separated value file “*.csv”. Please read
chapter "Result Files" later on.
Commands
Menu to "Load a Replayer File" for a short analysis of a recorded file. Menu to start and stop the exploring mode
"Explore Cell Culture" or an "Experiment". Menu to "Save Results".
Settings
This menu is also available as "Settings" dialog in the left control section of the main menu.
Help
Menu to view the online help and the "About" dialog.
Control Section
First dialog of the "Control Section" is the "Settings" dialog.
Lab Book
Dialog with three tabs for general information about the experiment. Fill in data referring to your experiment in
"Study" tab, "General" notes about the scientist and institution and free text in "Notes" tab. The "Labbook Settings"
will be stored in the Multiwell settings file *.mws.
Compounds
This dialog allows the definition of series of concentration and the assignment of the compounds to the wells.
There are two series of default compounds provided: The dilution factor in "Default 1:10" is ten, and in "Default 1:3"
the factor three. Create a user defined compound series by overwriting the given example in the window. Please
consider the comma separated syntax, otherwise you get a syntax error message.
Add a "Wash-Out Phase" via check box, if necessary.
To define a compound, please click onto the respective button. Assign the compound to one well by clicking on the
well in the plate beside, assign it to several wells by drawing a rectangle over the desired wells.
Type in the compound "ID", the "Name", the "Medium" and "Notes".
Add
up to 24 new compounds. Remove
assignment for all compounds
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compounds, delete all compounds
and clear the
Experimental Flow
Define the length of the "Test" and "Wash-in" phases of the experiment. Add a "Wash-In Phase" if necessary.
Instead of a fixed duration of time via "Timer" you can start and stop both experimental phases "Manual". In manual
mode the respective phase of the experiment runs until you stop the phase actively by clicking on the respective
icon in the "Experiment Control"
window.
Analyzer
Changes of "Analyzer Settings" effect all electrodes in one well. Please see chapter "Tutorial".
Tooltips are available and guide through the settings of the "Analyzer" dialogs.
It is useful to configure the "Analyzer Settings" when the "Explore" mode is running. All changes are directly sent to
the instruments, that's why you can directly see the effects in the data stream. Decide on the basis of the running
data, whether your actual settings fit or not.
The "Analyzer Settings" dialog has two tabs for the configuration of the analyzer instruments: "Heartbeat Detector"
and "Heartbeat Processing".
Heartbeat Detector Tab
The left side of the dialog allows to define the parameters for detecting the waveform of a heartbeat / field potential.
Select the "User-defined" or "Signal-defined" "Detection Threshold" with the numeric up-down boxes. Define the
"Threshold" and the "Duration" in ms. Define the "Minimum Rise Time" and the "Maximum Rise Time" in µs.
Select the "Detection Timing" via "Local min/max" or "Global min/max. "Global min/max" searches for the global
minimum and maximum within the whole search interval. "Local min/max" extracts the first minimum from the
second half of the search interval and the local maximum directly preceding this minimum.
Define the "Search Interval" and the "Dead Time" in ms.
Define the "Time Stamp Type" for the "Reporting" display from the drop-down menu.
The right side of the dialog allows the assignment to the wells, the "Well Selection".
Click the "Apply changes to all wells of that compound" button
and the following dialog appears.
Select the settings you want to apply via check boxes. The settings will be immediately applied to all wells of the
currently selected compound.
and the same dialog appears. Select the settings you want to
Click the "Apply changes to all wells" button
apply via check boxes. The settings will be immediately applied to all wells.
Use the arrow buttons for "Undo last changes" and "Redo last changes".
Heartbeat Processing Tab
The "Heartbeat Processing" allows to define the "Heartbeat Averager", the "Heartbeat 2D Analyzer" and the
"QT Detector".
The "Heartbeat Averager" averages the waveform of a signal in relation to each heartbeat in a time segment.
Display several recordings of an electrode channel in overlay and average the values. The pre and post time in
ms before and after a heartbeat can be selected via up-down box.
The "Heartbeat 2D Analyzer" calculates the latency between the different electrodes in one well. Define the time
span in ms from the drop-down menu in which the "Propagation Time" should be calculated. The action potential
runs over the heart cell culture or tissue or whole heart from the pacemaker to the peripheral regions of the
syncytium. The excitation propagation is measured in high spatial resolution by the electrodes detecting cardiac
field potentials and extracting local activation times. The propagation time is the longest possible period for passing
of the excitation from the first detected excited electrode to the last detected excited electrode of one cluster. If the
propagation time is too short, you miss data for the complete heartbeat. If the propagation time is too long, the next
heartbeat starts and the results are incorrect.
The "Min Cluster Channels" gives the minimum number of channels at which heartbeats have to be detected to
form a cluster. For example, if a syncytium covers all 12 electrodes of a well it could be 6 otherwise it should be
less than 6. This function is displayed in the lower part of the "Result Section", the display "2D Latency" map.
The "QT Detector" detects the QT-like interval in the averaged heartbeats. Define the parameters for the QT-like
interval: The level of the offset, the "Search Start Offset" and the "Search Stop Offset" in ms and the "Noise
Suppression" in ms from the up-down boxes. Select the "T Detection Target" from the drop-down menu.
The “T Detection Target” defines your preferred detection target. The “Global Extremum” describes the extremum
over the greatest integral. “Maximum” or “Minimum” define the respective maxima or minima. Please see the
following picture.
Data Acquisition
Please define the "Sampling Rate" in Hz from the drop-down box in the "Data Source Settings" dialog. It is possible
to sample each electrode with a sampling rate of up to 50 kHz. A sampling rate of at least five times the highest
frequency of the signal of interest is generally enough for a reasonable representation of the signals. For most
applications, 25 kHz will be fine. Be aware, that the provided possibility to use up to 288 channels with a sample
rate of up to 50 kHz needs high memory capacity.
Stimulation
The headstage of the Multiwell-MEA-System is equipped with an internal stimulator. Each electrode in each well
can be used for stimulation via Multiwell-Screen software. Until this status of development one stimulus pattern is
available which can be mirrored. Stimulate bipolar in "Voltage" or monopolar in "Current" mode. It is recommended
to start with the negative part of the bipolar stimulation pulse first.
The "Simulator Settings" dialog has three tabbed pages.
Stimulus Definition
"Stimulation Mode": Define the stimulation mode via radio buttons, "Voltage" or "Current".
"Stimulus Pattern": Choose the shape of the stimulation pattern via check box "Symmetric". Modulate the
amplitude of the rectangle pulse of "Phase 1" in mV and the duration in μs via up-down boxes. Modulate the
amplitude of the rectangle pulse of "Phase 2" in mV and the duration in μs via up-down boxes when creating
an asymmetric pulse. Define the inter stimulus interval "ISI" in ms via up-down box.
"Sync Out": Enable the sync out for the stimulus via check box "On". Define the "Extension" in µs via up-down box.
The sync out signal is available on digital out port 1 on the interface board.
The settings in the up-down boxes influence the complete stimulus pattern which is immediately displayed in the
window below.
Download the stimulation parameter settings to the internal stimulus generator with the "Set" button.
Stimulation Electrodes
Dialog to select the wells and the electrodes to which you want to apply the stimulus. The defined stimulation
pattern is indicated in red
and can be assigned to one of the electrodes, resulting in monopolar stimulation. In
voltage stimulation mode, the inverted pattern is available as a green icon to allow bipolar stimulation
Click on one of the icons and select the electrode you want to assign the pattern to.
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Delete the electrode selection for the selected stimulator with the "Clear" button
Undo or redo the last command with the arrow buttons. Apply the stimulation pattern to all wells with the same
compound or to all wells
button.
of the plate. Download the settings to the internal stimulator with the "Set"
"Stimulation Electrodes" in "Current" Mode
The stimulation in current mode is monopolar, the stimulation electrode is indicated in red.
Each group of 12 electrodes is controlled by the same two stimulators. For 72- and 96-well plates, this means that
groups of three or four wells, respectively, are assigned to the same stimulator pair. If more than two of these wells
are stimulated in current mode, two stimulation electrodes have to be assigned to the same stimulator. In this case,
the current output can not be guaranteed to match the amplitude defined in the stimulation pattern because the
current is distributed between the stimulation electrodes depending on their impedances.
That is why the connected well groups are marked in yellow in the background. Additionally the following "Warning"
is displayed.
Download the stimulation electrode settings to the internal stimulus generator with the "Set" button.
Schedule
Decide via radio button to start and stop the stimulation manual or via protocol. Enable or disable the respective
check boxes to apply the stimulation during the phases continuously or via "Timer". Define the "Stimulation
Duration" in the up-down box and select the time unit via drop-down box beside. Download the settings to the
internal stimulator with the "Set" button.
Recorder
Dialog to define the "File name" and the "Path" where you want to store the files. Select a "File name prefix", a "File
name core" and a "File name suffix" from the drop-down menu. Select or deselect the "Data steams" you are going
to record by clicking the check boxes.
For each compound different files are created:
Extension *.mwd: Raw data files in binary format (analog, digital and stimulation)
Extension *.mwr: XML-files with analysis results and analysis settings.
Extension *.mws: XML-files with general settings of the experiment.
Optionally: Extension *.mwc: Cutouts (Spike Cutouts or Heartbeat Averages) in binary format.
During an experiment, the names of all generated files are displayed in the status bar of the footer. Because of the
huge amount of data it is recommended to use a SSD data memory.
Report Sheet
Create a report sheet with the Multiwell-Screen software directly after running an experiment. Please define the
parameter in the "Report Sheet settings" dialog.
"General": Create the report file name and the path to the storing folder.
"Logo": Define the file name of the logo, if you want to insert a logo on the title page of the report sheet.
"Dose-Response Plots": Select the "Style of Dose-Response Plots" from the drop-down menu. Click the check box
"Invert Y-Axis for Slope" to invert the y-axis of the non normalized HB-slope parameter of the dose response plots.
"Results per Well": Select the parameter you want to include in the report sheet via check box.
Experiment Control
Second dialog of the "Control Section" is the "Experiment Control" option.
This dialog schedules your experiment. Before you can start an experiment, please fix the well plate in the
headstage. There are two ways to do so, either press the left touch pad on the headstage or click the "Insert"
button
"Eject"
in this dialog. To remove the wellplate again, press the right touch pad or click the
button.
If the plate is not detected by the software, please make sure that the contacts on the plate are clean.
You can define the plate type manually by pressing the “Select Plate Type...” button and selecting the appropriate
plate type from the drop-down menu.
Next step is running a test phase of the experiment without adding compounds and without recording. Use this
"Explore" phase to adjust the "Analyzer Settings" for preparing the experimental phase. Click the "Explore" button
to start the "Explore" mode and the symbol turns to "Running"
the symbol turns to "Stop"
. Stop the "Explore" mode and
.
In the current version of the Multiwell-Screen software, the "Stimulator" is not available. Please see the "Download"
section of our web site for software updates.
After checking the experimental parameters, start the experiment with "Go"
checks the disk space.
. First, the software
Make sure that enough disk space is available otherwise the program is stopped. Click "Go!" again.
The different phases of the experiment will run automatically one after the other through the single wells of the
plate. The process is stopped each time a new compound or another dilution of the compound has to be applied.
Start the next phase automatically on "Timer" or manual by clicking on the respective phase icon, if "Manual" is
selected in "Analyzer Settings".
The actual applied dilution and the phase of the experiment are highlighted in blue.
Multiwell Selection Panel
Third dialog of the "Control Section" is the "Multiwell Selection Panel". In "Cardio" mode the 2D Latency map
is displayed in each well.
This dialog allows the selection of the well from which data should be displayed. You can change your selection
while the program is running, so you can view all wells to control the experiment without stopping in between.
Click on the desired well to select it. The selected well will be highlighted
selection in the "Data Display Section" and in the "Result Section".
in light blue. See the effect of your
If no experiment is running, it is possible to exclude a well. Please click on the well with the right mouse button.
To reselect it again, double-click the well with the right mouse.
Important: The electrode channels of a deselected well will neither be displayed nor recorded!
Select the display mode from the drop down menu.
Temperature Control and Logfile
Last dialog of the "Control Section" is the "Temperature Control" and the "Logfile" available in two tabbed pages.
Temperature Control Tab
The headstage of the Multiwell-MEA is equipped with an internal temperature controller. That is, why the
"Temperature Control" starts automatically when the software is started. The temperature controller is not disabled
when the software is closed. Stop the temperature control by clicking the button "Disable". The drop-down menus
"Set Point" and "Range" will be active. Choose the temperature in "°C" or in "Fahrenheit" scale. Changes in
temperature set point will be directly sent to the device. The actual temperature is measured one time a second.
Choose the time span in minutes from the "Range" drop-down menu. Click the "Stop" button to pause the display,
the recording of the temperature data will go on. Temperature files are stored in "*.mws" data files.
Logfile Tab
The "Logfile" documents the ongoing experimental proceedings online. The "Logfile" data is stored in the "*.mwr"
results files.
Data Display Section
View your current data in this section. In "Cardio" mode, the data are presented in two formats in tabbed pages:
"Raw Data" and "Averages". In "Neuro" mode, there are three displays in the tabbed pages: "Raw Data", "Spike
Cutouts" and "PSTH Plots“ (Peri-stimulus time histogram). Please see chapter "Tutorial for Neuro Experiments "
for the neuro mode.
The upper display shows an overview of all electrodes of the well that was selected in the "Multiwell Control Panel"
dialog in the left "Control Section". The lower display shows the single view of one electrode.
Overview: Raw Data
The upper display shows the raw data of all electrodes, so you have a good overview about the activity in the
desired well.
See the identification of the selected well in the header of the dialog. The upper left electrode is selected by default.
Jump between the electrodes without stopping the experiment, the selected electrode will be highlighted in green
and viewed in detail in the "Single View". The red marker indicates the detection of a heartbeat or a spike in both
displays.
button and read the current value beside. The scaling of the y-axis is set
Click the "Adjust to signal Min/Max"
to the minimum and maximum of all samples in the well.
. Use the
Clicking the button "Zoom In" cuts the scaling in a half and "Zoom Out" doubles the scaling
up-down box to scale the time x-axis in each window of the overview. Deselect electrodes by clicking the "Exclude
Channel"
button first and then on the electrode. double-click the electrode to select it again.
Important: Data of excluded electrodes will not be recorded and stored!
One channel of the well has to be defined for analysis and display of the "Dose-Response" curve. Define this
electrode by clicking the "Select representative channel" button
green and framed in a blue frame.
. The analyzed channel will be highlighted in
Single View: Raw Data
The lower display shows the single view. The single view shows data of the selected electrode channel more
detailed.
In this example the raw data of the selected electrode No 21 is displayed. See the electrode ID in the left upper
edge of the display. Each field potential is described by its data trace and two additional set points: The red triangle
indicates the detection of a heartbeat, the blue diamonds mark the position of the maximum, the minimum and the
maximum slope of the Na peak.
Click the "Adjust to signal Min/Max"
all samples in the channel.
button. The scaling of the y-axis is set to the minimum and maximum of
. Additionally you can zoom
Zooming in cuts the scaling in a half and zooming out doubles the scaling
into a region of interest by moving the mouse from the left to the right while pressing the left mouse button. Move
the mouse the other way round for zooming out. Please read chapter "General Software Features".
Use the up-down box to scale the time axis of the single view display. Start and stop the data display independent
from the overview
stopped.
. In experimental mode the recording of the data will go on, even if the data display is
Overview: Averages
The "Average" display is for "Cardio" mode only and like the "Raw Data" separated in the upper overview and the
lower single view display.
In the "Overview" display you see the mean values of the data segment around all detected heartbeats.
See the identification of the selected well in the header of the dialog. The upper left electrode is selected by default.
Jump between the electrodes without stopping the experiment, the selected electrode will be highlighted in green
and viewed in detail in the "Single View".
button and read the current value beside. The scaling of the y-axis is set
Click the "Adjust to signal Min/Max"
to the minimum and maximum of all samples in the well.
Clicking the button "Zoom In" cuts the scaling in a half and "Zoom Out" doubles the scaling
.
One channel of the well has to be defined for analysis and display of the "Dose-Response" curve. Define this
electrode by clicking the "Select representative channel" button
green and framed in a blue frame.
. The analyzed channel will be highlighted in
Single View: Averages
In the "Overview" display you see the mean value of the data segment around all detected heartbeats. In the
"Single View" you see the mean value of the data segment around all detected heartbeats in this channel (the
black colored data trace) and additionally the standard deviation of each sample in red color.
In this example the averages of the selected electrode No 33 is displayed. See the electrode ID in the left upper
edge of the display.
Click the "Adjust to signal Min/Max"
all samples in the channel.
button. The scaling of the y-axis is set to the minimum and maximum of
. Additionally you can zoom
Zooming in cuts the scaling in a half and zooming out doubles the scaling
into a region of interest by moving the mouse from the left to the right while pressing the left mouse button. Move
the mouse the other way round for zooming out. Please read chapter "General Software Features".
Use the up-down box to scale the time axis of the single view display. Use the two up-down boxes to scale the time
axis of the single view display via "Pre" time before the averages and the "Post" time. Start and stop the data
display independent from the overview
if the data display is stopped.
. In experimental mode the recording of the data will go on, even
Single View: Overlay
Click the "Show Overlay" check box. Now the "Single View" display shows an overlay of the mean values of the
data segment around all detected heartbeats in this channel for the different concentrations of that experiment.
The traces of the different concentrations are color coded.
Result Section
View your current results in this section. Three types of result presentation are available in three dialogs:
"Dose-Response" and "2D Latency" and "Result per channel".
The upper display shows the "Dose-Response" dialog for each compound. The lower displays show the
"2D Latency" map and the "Result per channel" on the base of the selected well.
Dose-Response
The dose-response curve shows the variation of the physiological markers in relation to the concentration of the
compound.
The "Dose-Response" dialog looks different in dependency of the settings. All available compounds are displayed.
This example shows the field potential duration for the compounds 1 to 4.
Use the drop-down menus to organize the dose-response curves.
Choose the type of the curve from the "Parameter" drop-down menu: "FP Duration" describes the latency from
one extremum of the T-component of the heartbeat to the next extremum. "Peak-to-Peak" describes the interval
between the maximum and the minimum of a Na peak. "Slope" is the maximal slope of the Na peak. The
"RR Internal" is the time span between two heartbeats, measured from one maximum, that is the QRS-component
to the next maximum.
Choose the "Display Style": The "Mean" is calculated from the mean results of all wells of one compound.
This simple display format does not show the variability of the wells.
The "Mean + StdDev" shows additionally +/- 1 standard deviation of the mean and allows to estimate variability
between the wells but does not show outlier values.
The "Mean + Scatter" plot shows additionally to the mean the results of all concentrations of that well as own
points. Click on the points to make the results of this well but other concentration available. offers an easy way
to find outliers and to correct them, if necessary during offline analysis.
The "BoxPlot" shows the distribution of the results between the wells. You clearly see the wells which differ from
the median.
The Coefficient of Variation "CV" is a degree for the variability of a distribution. The "CV" is calculated from mean
divided by standard deviation and has no unit. You need at least two measurements to calculate the "CV", that is
why it is useless for parameters like QT interval or spike count. The "CV" displayed in this plot is the mean of the
"CV" of the concentrations in one single well.
See the variation of the physiological markers in the dose-response curve in absolute values "Raw Values"
or in "Normalize"
control section in %.
mode. Normalized results are calculated in relation to the respective
Experimental Control
Follow and control the process of the experiment in the left "Control Section" in the "Experimental Control" dialog.
The actual running phase is highlighted in blue.
Each time a new dilution of the compound is applicated, the "Pipette next dose" dialog appears.
Theoretical Background of Dose-Response Curve Calculation
The dose-response results from a well are computed using the activity on a single channel in the well. This
"representative channel" is automatically selected as the first channel in the well producing a heartbeat or spike.
As long as the experiment is not running, you can modify this selection by clicking the "Select representative
channel"
button in the "Raw Data Display", the "Averager Display" or the "Cutout Display" in Neuro mode.
The physiological parameters of this channel are collected during the experimental test period and the mean value
of the data is calculated. This mean value is the result for that well. The mean values of all wells with the same
compound in the same concentration gives the result for the respective dose. The sum of all concentrations build
the "Dose-Response" curve.
2D Latency Color Map
The 2D latency color map is for "Cardio" applications only. The map displays the propagation of a heartbeat from
the pacemaker to the outer regions of the cardiac cell culture. The isochrones are indicated by black lines. The
colors indicate the latency distribution over the well. Red color represents the pacemaker when using the color
map "jet inverse".
The electrodes in the well are symbolized by circles: Black circles indicate a detection of a heartbeat on this
electrode, white circles indicate no detection of a heartbeat, but the color map is completed by interpolation of data.
Below the map the current timestamp is displayed in hour:minute:second:millisecond.
Click the icon
to adjust the map parameters in the "Map Settings" dialog.
Map Settings Dialog: Data Tab
In the "Data" tab of the "Map Settings" dialog the interpolation parameter refers to the data, generating the map.
Default is the interpolation method "Nearest Neighbor". With the method of interpolation you can substitute missing
channels also. The program uses the same "Nearest Neighbor" method for interpolation: If you do not want to
substitute missing channels, click "Off". In the drop-down menu "Show Timestamp" you can choose the type of the
timestamp you want to be displayed below the color map.
Map Settings Dialog: Map Tab
In this dialog you have to define the settings for the "2D Latency" color map.
"Color Map": The typically used color map for cardiac issues is the "Jet Invers" map, but you can also choose
"Hot, Cool or Grey" maps. To remove the colors from the map, click "Off". The isochrones will remain only.
"Contour": The drop-down menu is for changing the color of the contour for the isochrones into "Black, Jet, Hot,
Cool and Grey". Or you can remove the lines with "Off". Adjust the number of the contour lines from the time scale
drop-down box beside, the smaller the time interval between the contour lines is chosen, the more isochrones are
available.
"Arrows": The feature is for indicating the main direction of the excitation. It is not implemented jet.
"Minimum / Maximum": You have to define the minimum and the maximum of the scale of the color map. That
means, you define the time frame in which the excitation pattern are displayed in the color map. Usually you start
with zero in the minimum, that is the pacemaker. The excitation pattern will run from the pacemaker over the map
to the maximum. The widest selectable time frame for the color map spans from 0 μs in the minimum to 10000 ms
in the maximum.
"Size (H x W)": Define the size of the map in pixel.
Interpolation: Interpolation is a mathematic method to calculate missing information inbetween two points of a line.
The bilinear interpolation uses four points of the nearest vicinity to calculate the interpolated point. The bicubic
method uses eight points to calculate the interpolated point. The interpolation method of the "nearest neighbor"
uses the mean value of four or five points to calculate the interpolated point.
"Show Electrodes": Decide via check box whether you want to display the black circles indicating the electrodes
in the map or not.
Result per Channel
Have a single view of one channel of the selected well in the "Result per Channel" display. See the electrode ID
in the middle over the display, the well ID in the header. Define the "Parameter" via drop-down menu: "Na Peak-topeak" or "RR-Interval". Start and stop the "Result per Channel" display independent from the compound overview
.
Zoom into accumulated data by moving the mouse from the left to the right while pressing the left mouse button.
Move the mouse the other way round for zooming out. Please read chapter "General Software Features".
Settings Files
You can store default parameters for the Multiwell-Screen software to have direct access to your preferred
experimental settings each time you start the software. This covers not only display settings, but also analyzer
settings, experiment settings, recorder settings and the content of all other dialogs accessible from the "Settings"
section of the main window.
The default settings are specific for each plate layout and application mode, “Cardio” or “Neuro” and are loaded
each time, a plate is inserted in the Multiwell headstage. To store the current configuration as default settings,
select “Save as Default Configuration” in the “Application” menu. To revert the current configuration to the default
configuration, select “Restore Default Configuration” from the “Application” menu.
If a set of different experiments is run regularly, it can be beneficial to have a default configuration containing the
common settings between the experiments, and a set of individual settings files, each storing the settings specific
for a single experiment. The current configuration can be stored in an external configuration file in XML format by
selecting “Save Configuration As...” from the “Application” menu. Pressing “Load Configuration” in the “Application”
menu allows the loading of either such a settings file in XML format or a *.mws file which is generated when an
experiment is started, storing the settings of this experiment.
When loading a configuration, you can select which parts of the settings should be loaded. For example, if you
want to keep your current "Analyzer" settings but load everything else from the settings file, disable the “Analyzer”
check box in this dialog. Loading a configuration is only possible if neither an experiment nor an exploration is
running.
If settings have been modified with respect to the default settings and the software attempts to load the default
settings again, either because the “Restore Default Configuration” entry in the “Application” menu has been
selected, or because a plate has been inserted, the software prompts the user on how to proceed before
overwriting the current configuration. This gives the user three options:
• Replace the default configuration with the current configuration by overwriting it.
• Store the current configuration in XML format.
• Discard the current configuration.
Result Files
When an experiment is finished, the analysis results can be exported to a "*.csv" file by selecting “Export Results”
from the “File” menu. This opens a dialog where the analysis parameters to be exported can be selected, as well
as the channels.
In “Cardio” mode, there are three tabbed pages from which you select the analysis parameters:
• Heartbeat
• Heartbeat 2D
• QT-related Interval
On each tab page, there are a number of parameters which can be selected or deselected. Clicking the “Export”
button on one of the pages will export these parameters to a "*.csv" file.
The tabs of the export dialogs are build analogical. On the left side of the dialog enable or disabled the check
boxes to choose the parameters you are interested in. Select the designated electrode channel on the MEA
electrode field and select the wells.
to select or deselect all electrodes of a well, click onto
Use the "Select all Electrodes in the Well" buttons
a single electrode to select or deselect one of them. Apply the stimulation pattern to all wells with the same
compound or to all wells
of the plate.
to export the file. These file can be opened, for example, in
Click the respective button
Excel. Please import the file to Excel, do not double-click on the "*.csv" file to open it, otherwise the parameters are
not separated in columns. Excel opens the "Text Import Wizard" dialog for the import of a "*.csv". Please click the
option "Delimited" for the original data type and click "Next".
Select option "Comma" in "Delimiters" window. Click "Finish" to import the file.
The "*.csv" file is opened. Now you can use it for further analysis.
In “Neuro” mode, the spike time stamps and the spike bursts can be exported.
In the “Well Selection” part of the dialog, select a well by a left mouse button click. In the “Well” part of the dialog,
you can select channels to export by clicking the grey squares. Selected channels are displayed in red.
Deselecting a channel can be done by clicking it again. Pressing the “Select all” button will select all channels
in the well, while pressing the “Deselect all” button will deselect all channels.
As in the “Analyzer” settings dialog, pressing the “Apply to compound” button will copy the channel selection of
the current well to all wells with the same compound. Pressing the “Apply to all wells” button will copy the channel
selection to all wells. The “Undo” button reverts the last action while the “Redo” button performs it again.
If no plate is inserted, results generated earlier during an experiment and stored in a file with analysis results with
the extension "*.mwr" can be loaded by pressing “Load Results” in the “Application” menu. These results of a
single compound are displayed in the dose-response plot and can be exported to a "Comma Separated File" with
"*.csv" extension as above via the “Export Results” entry in the “Application” menu.
Tutorial for Neuro Experiments
This tutorial explains step by step how to organize a neuro experiment with the Multiwell-Screen software.
The typical neuro experiment is arranged in different phases, but at first the experiment must be well prepared.
Please read the tutorial step by step.
To start the software, please click the "Multiwell-Screen" icon or select it from the program menu.
Before your first start, the Multiwell-Screen offers the general software determination for cardio or the neuro
applications. Select the desired application by clicking on the respective icon in the "Select your Application"
dialog. The selected mode will be stored as default. To change later from cardio to neuro application or vice versa,
please use the "File" menu, option "New".
Click the "Neuro Application" icon and the "Main Menu" window appears.
The main window in "Neuro" mode is build analogously to the main window in "Cardio" mode. The left "Control
Section" side consists of the same dialogs. The "Data Display Section" is different and has three tabbed pages:
"Raw Data", "Spike Cutouts" and "PSTH Plots“, that is the "Peri-stimulus time histogram". The "Result Section" on
the right side holds additionally to the "Dose-Response" curve the neuro application specific dialog "Spike Counts".
Most of the functions in "Neuro" mode are similar to them in "Cardio" mode. Please read the chapter "Software" for
detailed information. This tutorial explains neuro specific functions only.
Control Section
Configuration of the Experiment via "Settings" dialog.
Lab Book
Start the configuration of your experiment with the first "Settings" command "Lab Book".
Type in all information you need for this experiment in the "Study", "General" and "Notes" tabbed pages.
Compounds
Define the "Compounds & Dilution Series". Please read chapter " Compounds" for detailed information.
Experimental Flow
Define the "Experimental Flow". Please read chapter " Experimental Flow" for detailed information.
Analyzer
Analyzer Settings for Spike Detection and Spike Processing
Please read the chapter "Theoretical Background of Spike Detection" for more information about spike detection.
Tooltips are available and guide through the "Analyzer" dialogs.
Define the "Analyzer Settings" in the "Spike Detector" and "Spike Processing" tab. It is useful to configure the
"Analyzer Settings" when the "Explore" mode is running. All changes are directly send to the instruments, that's
why you can directly see the effects in the data stream. Decide on the basis of the running data, whether your
settings fit or not.
Spike Detector
The "Spike Detector" tab allows to set the "Detection type" from the drop-down menu. In dependency of the
detection type the dialog offers different options.
Detection Type: Threshold
Define the "Rising Edge" or "Falling Edge" in the "Edge Selection" window.
In the "Automatic Threshold Estimation" window you can select the standard deviation for the rising and falling
edge from the numeric up-down boxes. Apply these estimations to the currently selected well by pressing the
button "Estimate" or to all wells with the button "Estimate for all wells".
Use the up-down box "Number of Segments" to define, how many individual segments are used to estimate the
thresholds. The duration of each segment is defined by the “Baseline Duration”. The threshold is computed as the
minimum standard deviation over all segments, multiplied by either the “Rising Edge” or “Falling Edge” factor.
Using multiple short segments for the computation of the standard deviation instead of one long segment reduces
the risk that the standard deviation is strongly influenced by spike activity.
Select the "Dead Time" in µs in the "timer" window.
When using "Cutouts", please define the "Pre Trigger" and "Post Trigger" in µs from the numeric up-down boxes.
On the right side of the dialog the wellplate is displayed for the "Well Selection". As usual, the currently selected
well is highlighted with a blue circle.
Click the "Apply Changes to all Wells" button
and the following dialog appears "Select Settings to Apply".
Select the settings you want to apply via check boxes. The settings will be immediately applied to all wells of the
currently selected compound.
and the same dialog appears. Select the settings you want to
Click the "Apply changes to all wells" button
apply via check boxes. The settings will be immediately applied to all wells.
Use the arrow buttons for "Undo last changes" and "Redo last changes".
Detection Type: Slope
Changing the type of the detection for the spike detector to "Slope" the left side of the dialog will change
respectively.
In this case you have to define the parameters for the "Slope Detection". Please read chapter "Software".
Spike Processing
Tabbed page to adjust the "Spike Binning", the "Burst Detector" and the "Automatic Channel Selection".
Spike Binning
Use the up-down boxes in the "Spike Binning" window to define the time duration of the "Bin Width" in ms together
with the "Pre Trigger" and "Post Trigger".
Burst Detection
The “Burst Detection” option allows the detection and analysis of spike bursts. The spikes have to be detected first.
The quality of the burst analysis therefore depends on the accuracy of the spike detection.
The MaxInterval Method for detecting bursts
Find all bursts using the following algorithm:
Scan the spike train until an interspike interval is found that is less than or equal to Max. Interval.
While the interspike intervals are less than Max. End Interval, they are included in the burst.
If the interspike interval is more than Max. End Interval, the burst ends.
Merge all the bursts that are less than Min. Interval between Bursts apart.
Remove the bursts that have duration less than Min. Duration of Burst or have fewer spikes than Min. Number of
Spikes.
The detection parameters for bursts are shown in the following schema.
Parameter for burst detection:
Max. interval to start bursts: maximum interspike interval to start the burst.
Max. interval to end a burst: maximum interspike interval to end the burst.
Min. interval between bursts: minimum interspike interval between two bursts.
Min. duration of burst: minimum burst duration.
Min. number of spikes in burst: minimum number of spikes in a burst.
(Reference: Legendy C.R. and Salcman M. (1985): Bursts and recurrences of bursts in the spike trains of
spontaneously active striate cortex neurons. J. Neurophysiology, 53(4) : 926-939).
In relation to the "Burst Detection" parameter it is possible to select each of these parameters in the data display
window.
Automatic Channel Selection
This tool selects the representative channel for the calculation of the dose-response curve via "Max Spike Rate" or
via "Max Amplitude". Define the time span for the calculation interval with the up-down box "Duration in seconds.
Apply the selection to all wells by pressing the button "Run channel selection for all wells".
Data Acquisition
Please define the "Sampling Rate" in Hz from the drop-down box in the "Data Source Settings" dialog. " Data
Acquisition"
Multiwell Selection Panel
The only difference to the "Control Section" in "Cardio" mode is function of the display in the "Multiwell Selection
Panel". In "Cardio" mode you have an excitation map, in "Neuro" mode you have two opportunities, select the spike
count detection rate or the selected channel number from the drop-down menu "Display Style".
The channel activity is displayed in terms of spike count in a moving window. The higher the spike count rates, the
darker the color of the channel in the 2D map. Change the color of the map in the "Spike Count" display in the
"Map Settings" dialog.
Click "Selected Channel" to show the number of the channel which is automatically selected via spike detection.
Change the selection manually by clicking onto a different electrode in the data display.
Data Display Section
View your current data in this section. In "Neuro" mode, there are three displays in the tabbed pages: "Raw Data",
"Spike Cutouts" and "PSTH Plots“ (Peri-stimulus time histogram).
The upper display shows an overview of all electrodes of the well that was selected in the "Multiwell Selection
Panel" dialog in the left "Control Section". The lower display shows the single view of one electrode.
Spike Cutouts
The "Spike Cutouts" window is separated in the upper overview and the lower single view display.
In the "Overview" display you see the mean values of the data segment around all spike cutouts.
The "Spike Cutouts" display is specific for Neuro mode. Functions and tools are similar to the " Overview:
Averages" display in Cardio mode.
button and read the current value beside. The scaling of the y-axis is set
Click the "Adjust to signal Min/Max"
to the minimum and maximum of all samples in the well.
.
Clicking the button "Zoom In" cuts the scaling in a half and "Zoom Out" doubles the scaling
One channel of the well has to be defined for analysis and display of the "Dose-Response" curve. Define this
. The channel analyzed in the single view will
electrode by clicking the "Select representative channel" button
be highlighted in green and the channel with the blue frame will be used for the result analysis.
The screen is permanently updated. Data which can not be displayed for lack of space are available for the
calculation of the histogram anyway. Save an image of the screen by clicking the "Export"
icon.
Single View
The single view shows an overlay plot of the last cutouts of the selected channel. Define the "Time" scale in the
single view with the "Pre" and "Post" up-down boxes in ms. Select the number of the plotted cutouts via "Number
of Cutouts" up-down box.
PSTH Plots
Peri-stimulus time (PST) histograms display the distribution of spike times relative to the onset of each stimulus
presentation. They are often the most useful way to show temporal discharge patterns. Useful bin widths range
from 0.1 ms or less for brief stimuli such as up to 10 ms or more for stimuli lasting several seconds.
The neuronal response due to a single stimulation consists of one component, which is causally determined by
the pulse and of several components, which are not caused by the stimulation, spontaneous activity, for example.
If all components of the response have similar amplitudes as the stimulus induced component, it is not possible to
see this component. That is, why the Peri-Stimulus-Time-Histogram (PSTH) is calculated. The histogram is the
arithmetic mean over several identical stimulations and shows the time point, which is directly correlated to the
stimulus "Multiwell-Screen Software".
The screen is permanently updated. Data which can not be displayed for lack of space are available for the
calculation of the histogram anyway. Save an image of the screen by clicking the "Export"
icon.
The PSTH plot below was plotted during stimulation. The blue bar in the PSTH plot indicates the time stamp of the
stimulus.
Raw Data Section
The "Overview" display shows the raw data of all electrodes, so you have a good overview about the activity in the
desired well.
See the identification of the selected well in the header of the dialog. The upper left electrode is selected by default.
Jump between the electrodes without stopping the experiment, the selected electrode will be highlighted in green
and viewed in detail in the "Single View". The vertical red marker indicates the detection of a heartbeat or a spike in
both displays. A horizontal red bar above the spike markers indicates the detection and duration of a spike burst.
button and read the current value beside. The scaling of the y-axis is set
Click the "Adjust to signal Min/Max"
to the minimum and maximum of all samples in the well.
. Use the
Clicking the button "Zoom In" cuts the scaling in a half and "Zoom Out" doubles the scaling
up-down box to scale the time x-axis in each window of the overview. Deselect electrodes by clicking the "Exclude
Channel"
button first and then on the electrode. Click the electrode to select it again.
Important: Data of excluded electrodes will not be recorded and stored!
One channel of the well has to be defined for analysis and display of the "Dose-Response" curve. Define this
electrode by clicking the "Select representative channel" button
in green and framed in a blue frame.
. The analyzed channel will be highlighted
The following screenshot shows "Raw Data" display with the time stamps of stimulation indicated by the orange
triangle in the "Single View".
Single View: Raw Data
The lower display shows the single view. The single view shows data of the selected electrode channel more
detailed.
In this example the raw data of the selected electrode No 13 is displayed. See the electrode ID in the left upper
edge of the display. Set the threshold for the spike detection individually for each electrode. The detection events
are displayed in red for the upper detection point and blue for the lower detection point on the "Time" axis.
Click the "Adjust to signal Min/Max"
all samples in the channel.
button. The scaling of the y-axis is set to the minimum and maximum of
. Additionally you can zoom
Zooming in cuts the scaling in a half and zooming out doubles the scaling
into a region of interest by moving the mouse from the left to the right while pressing the left mouse button. Move
the mouse the other way round for zooming out. Please read chapter "General Software Features".
Use the up-down box to scale the time axis of the single view display. Start and stop the data display independent
from the overview
stopped.
. In experimental mode the recording of the data will go on, even if the data display is
Result Section
View your current results in this section. Two types of result presentation are available in two dialogs:
"Dose-Response" and "Spike Count".
The upper display shows the "Dose-Response" dialog for each compound. The lower displays show the
"2D Spike Count" map on the base of the selected well.
Dose-Response
The dose-response curve shows the variation of the physiological markers in relation to the concentration of the
compound. Please read more about "Theoretical Background of Dose-Response Curve Calculation " in chapter
"Software".
The "Dose-Response" dialog looks different in dependency of the settings. All available compounds are displayed.
This screenshot shows the "Normalized" "Mean" value of the only available parameter "Spike Count" for the
compounds 1 and 2.
Choose the "Display Style": The "Mean" is calculated from the mean results of all wells of one compound.
This simple display format does not show the variability of the wells.
See the variation of the physiological markers in the dose-response curve in absolute values "Raw Values"
or in "Normalize"
control section in %.
mode. Normalized results are calculated in relation to the respective
The "Mean + StdDev" shows additionally +/- 1 standard deviation of the mean and allows to estimate variability
between the wells but does not show runaway values.
The "Mean + Scatter" plot shows additionally to the mean the results of all concentrations of that well as own
points. Click on the points to make the results of this well but other concentration available. This display format
allows simply to find runaways to correct them, if necessary during offline analysis.
The "BoxPlot" shows the distribution of the results between the wells. You clearly see the wells which differ from
the median.
The Coefficient of Variation "CV" is a degree for the variability of a distribution. The "CV" is calculated from mean
divided by standard deviation and has no unit. You need at least two measurements to calculate the "CV", that is
why it is useless for parameters like spike count or QT interval in "Cardio" mode.
Spike Counts
Additionally to the "Dose-Response" curves the dialog "Spike Counts" is available in "Neuro" mode.
2D Spike Counts
The channel activity is displayed in terms of spike count in a moving window. The higher the spike count,
the darker the color of the channel in the 2D map. Spike counts are not interpolated between channels.
Below the map the current timestamp is displayed in hour, minute, second, and millisecond.
Modify the value in the “Width” numeric-up-down control to adjust the time range in which the spike counts are
computed. The value given in the “Update Interval” numeric-up-down control specifies the update rate of the spike
count maps. The mapping of spike counts to color can be adjusted in the “Map Tab” of the “Map Settings Dialog”
with the parameters “Minimum” (represented by white color) and “Maximum” (represented by black color). Values
outside this minimum-maximum range will be displayed either as white or black. "Click the icon to adjust the map
parameters in the "Map Settings" dialog.". Please read the respective chapter in "Software". Change the color of
the map for a better view.
See the "Experiment Control" dialog in the "Control Section" on the left side of the main menu. This dialog
schedules your experiment.
Prepare the wellplate you want to use carefully. Insert the wellplate in correct orientation in the Multiwell
headstage.
Before you can start an experiment, please fix the wellplate in the headstage. There are two ways to do so,
either press the left touch pad on the headstage or click the "Insert" button
To remove the wellplate again, press the right touch pad or click the "Eject"
Press the "Explore"
in this dialog.
button.
button.
It is useful to configure the "Analyzer Settings" when the "Explore" mode is running. All changes are directly send
to the instruments, that is why you can directly see the effects in the data stream. Decide on the basis of the
running data, whether your actual settings fit or not.
Changes of "Analyzer Settings" effect all electrodes in one well. The only exception is the "Spike Detector" in the
"Raw Data Display" in the "Neuro" modus, the threshold settings can be changed per channel. Please see "Raw
Data Section" in this chapter "Tutorial".
After passing all these preparation steps you can now start your experiment. Click the "Go"
button.
During the experiment the phases are repeated in dependency of the number of compounds.
Preliminary Phase
Control Phase
Pipetting of the first compound
Wash in
Test
Analysis
Pipetting of the next dilution of the compound
Wash in
Test
Analysis
Replayer Mode
The “Replayer” mode gives you the opportunity to re-run the analysis on your recorded Multiwell data files in order
to optimize the analysis parameters.
Please select first the application mode, "Cardio" or "Neuro" according to the file to be replayed.
Switch from “Measurement” to “Replayer” mode with the main menu “Application”, option “Switch to Replayer
Mode”.
The user interface „Experiment Control“ and corresponding commands in the main menu will change.
Load File
Please select a raw data file first. Load raw data files using the „Open“ or „Load Replayer File“ commands.
In the “Load Data File” dialog, press the upper "Open" button
"*.mwd".
to load a raw data file with the extension
If the folder containing the selected raw data file also contains a matching settings file with the extension "*.mws",
it will be automatically selected. This should be the case as long as all files generated in an experiment are kept
together in the same location. Otherwise, or if you want to load a different settings file, press the lower "Open"
to select a settings file with the extension "*.mws" or "*.xml" file. With the exception of the analyzer
button
settings, the contents of the settings file have to match the settings that were used during data acquisition.
Pressing the “OK” button will load the associated files or an error message appears if the settings file does not
match the data file.
The name of the file is displayed in the status bar of the footer.
Explore Mode
In "Explore" mode, the recorded data is displayed and analyzed in its original speed. Click on the "Explore" icon in
order to start or stop exploring. The data recorded during the test phase of this concentration will be played after
pressing the “Explore” button. Please use the drop-down menu to the right of the “Explore “ button to select one
of the substance doses contained in the data file, for example 300 pM. Just like in recording mode, the explore
function of the replayer enables you to modify analysis. You can directly see the effects on incoming raw data in
real time.
Use the "Explore" mode to select a representative channel for each well, which will later be used for dose response
results. This selection will be used for all doses. Keep the "Explore" mode running for at least 10 seconds in order
to automatically select representative channels.
Experiment Mode: Restart the Analysis of a complete File
Restart the analysis of the complete file using the „Go!“ command. The analysis runs through, one concentration
after the other until the experiment is stopped with the "Wash Out" phase.
Running a complete analysis will be performed as quickly as possible. In order to improve performance, raw data
will not be displayed in this mode neither in overview nor in single view. Please switch to "Averages" display to
observe data. Additionally the "2D Latency" maps in "Cardio" application and "Spike Count" maps in "Neuro"
application are disabled.
Save Results
After an experiment is finished and you are satisfied with the analysis results, press the “Save results after
replaying” button
files on the hard disk.
or use the main menu "Command" option to generate a report sheet and store the result
Using the "Save Results" command the Multiwell-Screen replayer data files results (*.mwr), settings (*.mws) and
cutout (*.mwc) will be saved to the directory defined in the recorder settings. File names are automatically created
according to the recorder settings. No existing file will be overwritten, file names will automatically be modified with
an increasing number. The file dialog which opens after clicking the "Save" icon, enables you to save a new report
file. Please select location and name for the new file. If you are not going to save a new report file, just close the
dialog.
General Software Features
The chapter "General Software Features" describes some of the Multiwell-Screen and Multiwell-Analyzer software
features on base of examples.
Numeric Up-Down Box
Adjust a value in the numeric up-down box either by clicking on the arrow buttons or by clicking into the window
and moving the mouse wheel. Turn the wheel forward and the level increases, turn the wheel backward and the
value decreases in fast steps. Use the arrow buttons for fine tuning the adjustment. The third possibility is a
replacement of the number in the window by overwriting it, if the value is predefined, for example.
Zoom In and Zoom Out
Zoom Buttons
Click the "Adjust to signal Min/Max" button. The scaling of the y-axis is set to the minimum and maximum of
all visible samples in the channel.
Click the "Zoom" buttons. Zooming in cuts the scaling of the respective axis in a half and zooming out
doubles the scaling.
Zoom by Mouse-click
Additionally to the zoom buttons you can freely zoom into a region of interest by moving the mouse inside a display
from the left to the right while pressing the left mouse button. Move the mouse from the right to the left for zooming
out.
Display Floating
Design your own display with the floating feature. Decouple a dialog display of your choice and place it wherever
you want.
Double-click the floating window to a sent it back to its default place.
Tooltips
In some dialogs tooltips are available to guide through the respective settings.
MEA Filter Configuration
The MEA Filter Configuration software is an add on program for changing the hardware filter settings by the
software. No firmware update is necessary. You can operate the software with Multiwell-MEA-Systems and
MEA2100-Systems.
Please install the MEA Filter Configuration software by clicking the “MeaFilterConfig.exe” file. Follow the
installation guide as usual.
Main Window of the MEA Filter Configuration program
The MEA Filter Configuration program for changing the hardware filter setting of the MEA2100-System is selfexplanatory via tool tips. It is possible to control each headstage separately.
Define the “MEA Devices” from the drop-down box if two headstages or headstages with two MEAs are available.
Press "Refresh" button to update the settings. The actual settings for “High and Low Pass Filter” for the selected
headstage are displayed. If the actual settings are not on the drop-down list, “Custom” will be displayed.
Select the order of the filter from the first drop-down box. The message “The filter order determines the steepness
of the filter” is shown. The higher the order, the steeper the cut off.
Select the cut off frequency of the filter from the second drop-down box. The message “The cut off frequency of the
filter. The amplitude is reduced by 3 db at this frequency. A filter with Bessel characteristic is used.” is shown.
The option “Custom” can not be selected. If you need a frequency which is not available on the list, please contact
[email protected].
The selected settings are automatically downloaded to the MEA2100 device as soon as you select them. If the
“Make Permanent” button is not used, the device will be back to the last saved settings after a reset.
Click the button “Make Permanent” to store the selected filter settings permanently.
Click the button “Defaults” to refresh the factory default settings.
Theoretical Background
Cardiac Action Potential
Over the last 40 years, non-invasive extracellular recording has developed into a widely used standard method.
The semi-permeable lipid bilayer cell membrane separates different ion concentrations (charges) on the inner and
outer side of the membrane. Conventional methods measure the membrane potential that results from the
electrochemical gradient directly with an intracellular electrode. When ion channels are opened due to chemical
or electrical stimulation, the corresponding ions are moving along their electrochemical gradient. In other words,
the resistance of the membrane is lowered, resulting in an inward or outward flow of ions, measured as a
transmembrane current. The extracellular space is conductive as well, and though the resistance is very low, it is
not zero. According to Ohm's law (U=R*I), the extracellular current results in a small voltage that can be measured
with extracellular electrodes. Extracellular signals are smaller than transmembrane potentials, depending on the
distance of the signal source to the electrode. Extracellular signal amplitudes decrease with increasing distance of
the signal source to the electrode. Therefore, a close interface between electrode and cell membrane is very
important for a high signal-to noise ratio.
The transmembrane current and the extracellular field potential follow the same time course. The field potential is
roughly equal to the first negative derivative of the transmembrane potential, thus revealing an information that is
comparable to conventional methods. This has been shown for different types of signals derived from neuronal
preparations as well as cardiac preparations. This convenient method is used by Multiwell-Screen. Simultaneous
recordings of action potentials (with intracellular electrodes) and field potentials (with extracellular electrodes) have
shown that there is a linear relationship between the rise time of the cardiac action potential (AP) and field potential
(FP) as well as between AP and FP duration. The contribution of different ionic transmembrane currents can be
identified in the shape of the FP waveform as well, as shown in the following picture. The correlation between the
waveform components and the ion channel activities was shown by using ion channel blockers or depleting the
medium of the respective ions. You can clearly see the rapid component of the depolarizing sodium current and
the slow calcium current. The delayed rectifying K+ current (IKr) is represented either by a positive or negative
peak. The polarity of the peak depends on several parameters, for example, the proximity of the cell layer to the
measuring electrode, and cannot be predicted, which does not matter for this assay. The field potential duration
corresponds to the action potential duration, which can be correlated to a QT-related interval in an
electrocardiogram. It is measured from minimum of the Na+ peak to the maximum / minimum of the IKr current
peak.
The delayed rectifying K+ current (IKr) is represented either by a positive or negative peak. The polarity of the peak
depends on several parameters, for example, the proximity of the cell layer to the measuring electrode, and cannot
be predicted, which does not matter for this assay. The field potential duration corresponds to the action potential
duration, which can be correlated to a QT-related interval in an electrocardiogram. It is measured from minimum of
the Na+ peak to the maximum / minimum of the IKr current peak.
(Reference: "Estimation of Action Potential Changes from Field Potential Recordings in Multicellular Mouse
Cardiac Myocyte Cultures", Marcel D. Halbach et al., Cell Physiol Biochem 2003;13:271284)
Theoretical Background of Spike Detection
The "Spike Detector" is used for extracting spikes from the electrode raw data. There are two ways of detecting
and extracting spikes with Multiwell Screen software. One method uses a threshold, the other recognizes the
shape that is, the amplitude and slope of a spike curve. Both methods regard either the rise or the fall of a curve,
depending on your settings. Waveforms that meet the requirements defined by either of the two methods are cut
out from the raw data. The size of the cutout, that is, the time length before and after the spike detection event,
can be chosen by the user. It is also possible to extract only the timestamps.
The "Threshold" method, the spike detection level is especially useful if the overall signal is steady and the spikes
appear approximately on the same height. If you have local field potentials or a high noise level, you will miss
spikes or detect noise signals with the "Threshold" method. In this case, the "Slope" method is more appropriate.
The following illustrations show how the two methods work.
Technical Specifications
General characteristics
Operating temperature
Storage temperature
Relative humidity
10 °C to 50 °C
0 °C to 50 °C
10 % to 85 %, non-condensing
Headstage
Dimensions (W x D x H)
Weight
Well plate contacting unit
302 mm x 166 mm x 90 mm
+/- 4000 g
Motorized contacting mechanism
Amplifier
Number of analog recording channels
Data resolution
Input voltage range
Bandwidth
Sampling frequency per channel
288
24 bit
+/- 500 mV
0.1 Hz to 10 kHz (modifiable via MEA Filter Config software);
default settings: 1 Hz to 3.5 kHz
50 kHz or lower, software controlled
Integrated Stimulator
Current stimulation
Voltage stimulation
max. +/- 500 μA
max. +/- 10 V
Integrated Heating Element
Temperature sensor type
Accuracy of regulation
Pt 100 with four wire connection
+/- 0.1 °C
MCS Interface Board
Dimensions (W x D x H)
Weight
4 x Digital Out
4 x Digital In
16 bit Digital In- Out
1 x Audio Out
2 x Analog In
Analog In
1 x Ground
1 x DSP connector
USB 3.0 Port
250 mm x 83 mm x 25 mm
300 g
Lemo connector, EPL.00.250.NTN
Lemo connector, EPL.00.250 NTN
68-pin MCS standard connector
Stereo jack 3.5 mm, FFA.00.250 CTAC29
Lemo connector, EPL.00.250 NTN
20-pin connector (2.54 mm grid), dual row standard IDC
Common jack 4 mm, banana plug
JTAG
USB 3.0 cable
Power Supply
Power Supply
100 - 240 V input range
Software
Operating System
Data Acquisition
and Analysis Software
Analysis Software
Windows ® 8.1 (64 bit)
Multiwell-Screen, Version 1.3.2
Multiwell-Analyzer, Version 0.1.4
Contact Information
Local retailer
Please see the list of official MCS distributors on the MCS web site.
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